Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked

Origeneb

pGilda

HIS3

ApR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

Origeneb

pDD

HIS3

KmR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

R. Hopkinsa

Control LexA‐fused baits

pEG202‐Krev1

HIS3

ApR

A negative control for activation and positive control for interaction with Krit1 and RalGDS

I. Serebriiskiia

pEG202‐Krit1

HIS3

ApR

A moderate positive control for activation

I. Serebriiskiia

pRFHM1 (control)

HIS3

ApR

The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a
negative control for activation and interaction assays, and as a positive control for repression assay.

Origeneb

pSH17‐4 (control)

HIS3

ApR

GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.

Origeneb

Plasmid name (reading frames)

Yeast selection

E. coli selection

Comment/descriptionc

cI‐Fusion plasmid

pGLS23d (A, B)

HIS5

CmR

ADH1 promoter expresses cI followed by polylinker

Control cI‐fused baits

pGKS3‐Krev

HIS5

CmR

Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.

pGLS22‐Ras

HIS5

CmR

Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Rafd.

pGLS22‐EE12345L

HIS5

CmR

Expresses cI‐EE12345L fusion protein. Use as positive control for activation assay.

Name

Relevant genotype

Number of operators

Comment/description

Contact information

PRT50

MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2

6 lexA, 3 cI

Provides the most sensitive LexA‐responsive LEU2 reporter

I. Serebriiskiie

PRT475

MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2

4 lexA, 3 cI

Mating partner for PRT50 strain; can be also used as a reporter strain itself.

Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked

Origeneb

pGilda

HIS3

ApR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

Origeneb

pDD

HIS3

KmR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

R. Hopkinsa

Control LexA‐fused baits

pEG202‐Krev1

HIS3

ApR

A negative control for activation and positive control for interaction with Krit1 and RalGDS

I. Serebriiskiia

pEG202‐Krit1

HIS3

ApR

A moderate positive control for activation

I. Serebriiskiia

pRFHM1 (control)

HIS3

ApR

The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a
negative control for activation and interaction assays, and as a positive control for repression assay.

Origeneb

pSH17‐4 (control)

HIS3

ApR

GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.

Origeneb

Plasmid name (reading frames)

Yeast selection

E. coli selection

Comment/descriptionc

cI‐Fusion plasmid

pGLS23d (A, B)

HIS5

CmR

ADH1 promoter expresses cI followed by polylinker

Control cI‐fused baits

pGKS3‐Krev

HIS5

CmR

Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.

pGLS22‐Ras

HIS5

CmR

Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Rafd.

pGLS22‐EE12345L

HIS5

CmR

Expresses cI‐EE12345L fusion protein. Use as positive control for activation assay.

Name

Relevant genotype

Number of operators

Comment/description

Contact information

PRT50

MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2

6 lexA, 3 cI

Provides the most sensitive LexA‐responsive LEU2 reporter

I. Serebriiskiie

PRT475

MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2

4 lexA, 3 cI

Mating partner for PRT50 strain; can be also used as a reporter strain itself.

I. Serebriiskiie

cContact Ilya Serebriiskii at

dControl plasmids are cloned in a pGLS22 background; pGLS22 is identical to pGLS23 except for an extra EcoRI site outside the polylinker.

Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked

Origeneb

pGilda

HIS3

ApR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

Origeneb

pDD

HIS3

KmR

GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous
presence of the bait is toxic to yeast

R. Hopkinsa

Control LexA‐fused baits

pEG202‐Krev1

HIS3

ApR

A negative control for activation and positive control for interaction with Krit1 and RalGDS

I. Serebriiskiia

pEG202‐Krit1

HIS3

ApR

A moderate positive control for activation

I. Serebriiskiia

pRFHM1 (control)

HIS3

ApR

The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a
negative control for activation and interaction assays, and as a positive control for repression assay.

Origeneb

pSH17‐4 (control)

HIS3

ApR

GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.

Origeneb

Plasmid name (reading frames)

Yeast selection

E. coli selection

Comment/descriptionc

cI‐Fusion plasmid

pGLS23d (A, B)

HIS5

CmR

ADH1 promoter expresses cI followed by polylinker

Control cI‐fused baits

pGKS3‐Krev

HIS5

CmR

Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.

pGLS22‐Ras

HIS5

CmR

Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Rafd.

pGLS22‐EE12345L

HIS5

CmR

Expresses cI‐EE12345L fusion protein. Use as positive control for activation assay.

Name

Relevant genotype

Number of operators

Comment/description

Contact information

PRT50

MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2

6 lexA, 3 cI

Provides the most sensitive LexA‐responsive LEU2 reporter

I. Serebriiskiie

PRT475

MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2

4 lexA, 3 cI

Mating partner for PRT50 strain; can be also used as a reporter strain itself.

I. Serebriiskiie

eContact at
.

Basic Protocol 2: Preparation of Baits and Library: Transforming and Characterizing the Library